A Distinct Mechanism to Achieve Efficient Signal Recognition Particle (SRP)-SRP Receptor Interaction by the Chloroplast SRP Pathway

Cotranslational protein targeting by the signal recognition particle (SRP) requires the SRP RNA, which accelerates the interaction between the SRP and SRP receptor 200-fold. This otherwise universally conserved SRP RNA is missing in the chloroplast SRP (cpSRP) pathway. Instead, the cpSRP and cpSRP receptor (cpFtsY) by themselves can interact 200-fold faster than their bacterial homologues. Here, cross-complementation analyses revealed the molecular origin underlying their efficient interaction. We found that cpFtsY is 5- to 10-fold more efficient than Escherichia coli FtsY at interacting with the GTPase domain of SRP from both chloroplast and bacteria, suggesting that cpFtsY is preorganized into a conformation more conducive to complex formation. Furthermore, the cargo-binding M-domain of cpSRP provides an additional 100-fold acceleration for the interaction between the chloroplast GTPases, functionally mimicking the effect of the SRP RNA in the cotranslational targeting pathway. The stimulatory effect of the SRP RNA or the M-domain of cpSRP is specific to the homologous SRP receptor in each pathway. These results strongly suggest that the M-domain of SRP actively communicates with the SRP and SR GTPases and that the cytosolic and chloroplast SRP pathways have evolved distinct molecular mechanisms (RNA vs. protein) to mediate this communication

Transient tether between the SRP RNA and SRP receptor ensures efficient cargo delivery during cotranslational protein targeting

Kinetic control of macromolecular interactions plays key roles in biological regulation. An example of such control occurs in cotranslational protein targeting by the signal recognition particle (SRP), during which the SRP RNA and the cargo both accelerate complex assembly between the SRP and SRP receptor FtsY 10^2-fold. The molecular mechanism underlying these rate accelerations was unclear. Here we show that a highly conserved basic residue, Lys399, on the lateral surface of FtsY provides a novel RNA tetraloop receptor to mediate the SRP RNA- and cargo-induced acceleration of SRP–FtsY complex assembly. We propose that the SRP RNA, by using its tetraloop to interact with FtsY–Lys399, provides a transient tether to stabilize the early stage and transition state of complex formation; this accelerates the assembly of a stable SRP–FtsY complex and allows the loading of cargo to be efficiently coupled to its membrane delivery. The use of a transient tether to increase the lifetime of collisional intermediates and reduce the dimension of diffusional search represents a novel and effective mechanism to accelerate macromolecular interactions

Concerted Complex Assembly and GTPase Activation in the Chloroplast Signal Recognition Particle

The universally conserved signal recognition particle (SRP) and SRP receptor (SR) mediate the cotranslational targeting of proteins to cellular membranes. In contrast, a unique chloroplast SRP in green plants is primarily dedicated to the post-translational targeting of light harvesting chlorophyll a/b binding (LHC) proteins. In both pathways, dimerization and activation between the SRP and SR GTPases mediate the delivery of cargo; whether and how the GTPase cycle in each system adapts to its distinct substrate proteins were unclear. Here, we show that interactions at the active site essential for GTPase activation in the chloroplast SRP and SR play key roles in the assembly of the GTPase complex. In contrast to their cytosolic homologues, GTPase activation in the chloroplast SRP–SR complex contributes marginally to the targeting of LHC proteins. These results demonstrate that complex assembly and GTPase activation are highly coupled in the chloroplast SRP and SR and suggest that the chloroplast GTPases may forego the GTPase activation step as a key regulatory point. These features may reflect adaptations of the chloroplast SRP to the delivery of their unique substrate protein

Strainrange partitioning: A total strain range version

Procedures are presented for expressing the Strainrange Partitioning (SRP) method for creep fatigue life prediction in terms of total strain range. Inelastic and elastic strain-range - life relations are summed to give total strain-range - life relations. The life components due to inelastic strains are dealt with using conventional SRP procedures while the life components due to elastic strains are expressed as families of time-dependent terms for each type of SRP cycle. Cyclic constitutive material behavior plays an important role in establishing the elastic strain-range - life relations as well as the partitioning of the inelastic strains. To apply the approach, however, it is not necessary to have to determine the magnitude of the inelastic strain range. The total strain SRP approach is evaluated and verified using two nickel base superalloys, AF2-1DA and Rene 95. Excellent agreement is demonstrated between observed and predicted cyclic lifetimes with 70 to 80 percent of the predicted lives falling within factors of two of the observed lives. The total strain-range SRP approach should be of considerable practical value to designers who are faced with creep-fatigue problems for which the inelastic strains cannot be calculated with sufficient accuracy to make reliable life predictions by the conventional inelastic strain range SRP approach

Efficient Interaction between Two GTPases Allows the Chloroplast SRP Pathway to Bypass the Requirement for an SRP RNA

Cotranslational protein targeting to membranes is regulated by two GTPases in the signal recognition particle (SRP) and the SRP receptor; association between the two GTPases is slow and is accelerated 400-fold by the SRP RNA. Intriguingly, the otherwise universally conserved SRP RNA is missing in a novel chloroplast SRP pathway. We found that even in the absence of an SRP RNA, the chloroplast SRP and receptor GTPases can interact efficiently with one another; the kinetics of interaction between the chloroplast GTPases is 400-fold faster than their bacterial homologues, and matches the rate at which the bacterial SRP and receptor interact with the help of SRP RNA. Biochemical analyses further suggest that the chloroplast SRP receptor is pre-organized in a conformation that allows optimal interaction with its binding partner, so that conformational changes during complex formation are minimized. Our results highlight intriguing differences between the classical and chloroplast SRP and SRP receptor GTPases, and help explain how the chloroplast SRP pathway can mediate efficient targeting of proteins to the thylakoid membrane in the absence of the SRP RNA, which plays an indispensable role in all the other SRP pathways

Semantic Robot Programming for Goal-Directed Manipulation in Cluttered Scenes

We present the Semantic Robot Programming (SRP) paradigm as a convergence of robot programming by demonstration and semantic mapping. In SRP, a user can directly program a robot manipulator by demonstrating a snapshot of their intended goal scene in workspace. The robot then parses this goal as a scene graph comprised of object poses and inter-object relations, assuming known object geometries. Task and motion planning is then used to realize the user's goal from an arbitrary initial scene configuration. Even when faced with different initial scene configurations, SRP enables the robot to seamlessly adapt to reach the user's demonstrated goal. For scene perception, we propose the Discriminatively-Informed Generative Estimation of Scenes and Transforms (DIGEST) method to infer the initial and goal states of the world from RGBD images. The efficacy of SRP with DIGEST perception is demonstrated for the task of tray-setting with a Michigan Progress Fetch robot. Scene perception and task execution are evaluated with a public household occlusion dataset and our cluttered scene dataset.Comment: published in ICRA 201

Import of honeybee prepromelittin into the endoplasmic reticulum

Honeybee prepromelittin is correctly processed and imported by dog pancreas microsomes. Insertion of prepromelittin into microsomal membranes, as assayed by signal sequence removal, does not depend on signal recognition particle (SRP) and docking protein. We addressed the question as to how prepromelittin bypasses the SRP/docking protein system. Hybrid proteins between prepromelittin, or carboxy-terminally truncated derivatives, and the cytoplasmic protein dihydrofolate reductase from mouse were constructed. These hybrid proteins were analysed for membrane insertion and sequestration into microsomes. The results suggest the following: (i) The signal sequence of prepromelittin is capable of interacting with the SRP/docking protein system, but this interaction is not mandatory for membrane insertion; this is related to the small size of prepromelittin. (ii) In prepromelittin a cluster of negatively charged amino acids must be balanced by a cluster of positively charged amino acids in order to allow membrane insertion. (iii) In general, a signal sequence can be sufficient to mediate membrane insertion independently of SRP and docking protein in the case of short precursor proteins; however, the presence and distribution of charged amino acids within the mature part of these precursors can play distinct roles

The strainrange partitioning behavior of an advanced gas turbine disk alloy, AF2-1DA

The low-cycle, creep-fatigue characteristics of the advanced gas turbine disk alloy, AF2-1DA have been determined at 1400 F and are presented in terms of the method of strainrange partitioning (SRP). The mean stresses which develop in the PC and CP type SRP cycles at the lowest inelastic strainrange were observed to influence the cyclic lives to a greater extent than the creep effects and hence interfered with a conventional interpretation of the results by SRP. A procedure is proposed for dealing with the mean stress effects on life which is compatible with SRP